The 3′ untranslated region (UTR) of the POLD1 luciferase reporter plasmid (pHS-AVC-LW2301) was obtained from SyngenTech (Beijing, China). For the assessment of CTCF function on POLD1 promoter activity, genomic fragments harboring putative CTCF-binding sites in the human POLD1 promoter (∼1.8 kb upstream of the translation start site) were subcloned into the pGL4-Luc reporter vector (Promega) using Infusion 2.0 Dry-Down PCR cloning kit (Clontech, Shanghai, China). Promoter activity was further validated by mutation of the putative CTCF-binding site on the promoter at −1,015 to −997 or −625 to −607 by selecting these sites. For the luciferase reporter assay, 293T cells were seeded in a 24-well plate and incubated for 24 h before transfection. Subsequently, luciferase constructs, POLD1, and POLD1 + CTCF were co-transfected into 293T cells using Lipofectamine 3000. Cells were collected at 48 h after transfection and measured using the Dual-Luciferase Reporter System (Promega, WI, United States), according to manufacturer’s protocols. Three independent experiments were performed, and data were presented as mean ± SD.
Pgl4 luc reporter vector
Manufactured by Promega
Sourced in China
The PGL4-Luc reporter vector is a plasmid designed for the expression and measurement of firefly luciferase, a widely used reporter protein. The vector contains the firefly luciferase gene under the control of a promoter, allowing for the quantification of transcriptional activity in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Infusion 2.0 dry down pcr cloning kit, by Takara Bio (3 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Fugene, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
3 protocols using pgl4 luc reporter vector
1
POLD1 Promoter Regulation by CTCF
2
Assessing IFITM1 Promoter Activity
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For the assessment of IFITM1 promoter activity, the genomic fragments harboring the putative PITX2-binding sites in human IFITM1 promoter (~1.8 kb upstream of the translation start site), were subcloned into the pGL4-Luc reporter vector (Promega, Madison, WI) using Infusion 2.0 Dry-Down PCR cloning kit (Clontech, Shanghai, China). Promoter activity was further validated by mutation of the putative PITX2-binding site on the promoter at –406/–402 by replacing AATCC with AcgaC using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). For reporter assay, 0.5 μg reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected with pPM-His-PITX2 or pPMHis vector into HeLa cells using FUGENE (Promega). Forty-eight hours later, cells were treated for 4 hours with 20 U/mL of hIFNα, followed by luciferase activity measurements using a dual luciferase reporter assay kit (Promega).
3
Assessing HMOX1 Promoter Activity
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For the assessment of HMOX1 promoter activity, the genomic fragments harboring the putative KLF14-binding sites in human HMOX1 promoter (~1.8 kb upstream of the translation start site) were subcloned into the pGL4-Luc reporter vector (Promega) using Infusion 2.0 Dry-Down PCR cloning kit (Clontech, Shanghai, China). Promoter activity was further validated by mutation of the putative KLF14-binding site on the promoter at -406/-402 by replacing CACCC with CAaaa using the QuikChange Site-Directed Mutagenesis Kit (Agilent). For reporter assay, 0.5 μg reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected with pcDNA3.1+-DDK-KLF14 or pcDNA3.1+ vector into HeLa cells using FUGENE. 48 h later, cells were treated for 15 min with 1 mmol/L of H 2 O 2 , 30 mmol/L of diamide or diamide plus 20 μmol/L of diphenyleneiodonium chloride, followed by luciferase activity measurements using a dual luciferase reporter assay kit (Promega).
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